A fundamental unanswered question is whether the ability of master regulators of lineage commitment to control disparate fate decisions results primarily from unique functional capabilities or from the cellular context in which they are expressed. ThPOK and LRF are related members of the POK family of transcription factors, with essential roles at distinct stages of hematopoiesis. Thus ThPOK controls CD4 T cell commitment, while LRF controls B cell commitment. These factors also play critical roles at other stages of hematopoiesis, including for hematopoietic stem cell maintenance (see preliminary results). Despite their multiple critical roles, the mechanism/s underlying specific functions of these factors remains to be fully elucidated, including even the fundamental question of whether their distinct roles result primarily from unique functional capabilities or frm expression pattern differences. Here we propose to elucidate these questions according to two specific aims: Aim 1. Do ThPOK and LRF mediate inherently distinct functions during hematopoiesis? Non- redundant roles of ThPOK and LRF may reflect inherent functional differences, dictated by their distinct amino acid sequences, or alternative expression patterns. To resolve this question we have generated novel knockin mice in which ThPOK coding exons are precisely replaced by those of LRF (LRF knockin, or LKI mice). LKI mice have been engineered to maintain both DNA- and mRNA-dependent control mechanisms of gene expression, as all DNA cis regulatory elements as well as 5' and 3' untranslated (UT) mRNA regions are derived from the ThPOK locus. Therefore, the LKI allele supports precisely the same level and kinetics of gene expression as the wt ThPOK allele. We will use homozygous LKI/LKI mice to assess whether LRF can rescue defects in T cell development and early hematopoiesis in the absence of ThPOK, in order to definitively establish whether LRF and ThPOK coding sequences encode distinct or redundant functions. We present preliminary evidence that strongly suggests functional specialization. Aim 2. Assessing functional specificity of ThPOK BTB domain using a chimeric gene approach. If ThPOK is functionally distinct from LRF, specialized functions are likely to be encoded at least in part by their BTB/POZ domains, which are critical for dimerization with nuclear co-repressors and histone deacetylases. Despite the essential roles of the ThPOK and LRF BTB domains, their functional specificity/redundancy has never been examined in vivo. Here we will use a knockin approach to selectively replace the BTB domain of ThPOK with that of LRF, or vice versa, and test whether the resulting chimeric factors can restore thymic development of CD4 and iNKT cells in the absence of wt ThPOK. This will establish whether the ThPOK BTB domain is necessary and/or sufficient to replicate the essential role of ThPOK in these processes. These mice will also be valuable for dissecting the specific requirement for the ThPOK BTB domain in other aspects of hematopoiesis in which ThPOK has been implicated, including HSC maintenance and myeloid development.